Lysosomal storage diseases (LSDs) result from the absence or reduced activity of specific enzymes within the lysosomes of a cell. A large number of these enzymes have been identified and correlated with their related diseases. Once the missing or deficient enzyme has been identified, treatment can be reduced to the problem of delivering replacement enzyme (drug) to a patient's affected tissues. Within cells, the effect of the missing enzyme can be seen as an accumulation of undegraded “storage material” within the intracellular lysosome. This build-up causes lysosomes to swell and malfunction, resulting in cellular and tissue damage. As lysosomal storage diseases typically have a genetic etiology, many tissues will lack the enzyme in question. However, different tissues suffer the absence of the same enzyme differently. How adversely a tissue will be affected is determined, to some extent, by the degree to which that tissue generates the substrate of the missing enzyme. The types of tissue most burdened by storage, in turn, dictate how the drug should be administered to the patient. While intravenous enzyme replacement therapy (ERT) is beneficial for LSDs (e.g. MPS I, MPS II), means for enhancing the delivery of the therapeutic enzyme to the lysosome in such diseases would be advantageous in terms of reduced cost and increased therapeutic efficacy.
In addition, the blood-brain barrier (BBB) blocks the free transfer of many agents from blood to brain. For this reason, LSDs that present with significant neurological affect (e.g. MPS III, MLD, GM1) are not expected to be as responsive to intravenous ERT. For such diseases, a method of delivering the enzyme across the BBB and into the lysosomes of the affected cells would be highly desirable.
In the early 1980's, melanotransferrin (MTf) or p97 was identified as an oncofetal antigen that was either not expressed, or only slightly expressed in normal tissues, but was found in much larger amounts in neoplastic cells (especially malignant melanoma cells) and fetal tissues (Woodbury, et al., P.N.A.S. USA, 77:2183-2187 (1980)). More recently, there have been additional reports of human MTf being identified in normal tissues, including sweat gland ducts, liver endothelial cells and the endothelium and reactive microglia of the brain (Jefferies, et al., Brain Res., 712:122-126 (1996); and Rothenberger, et al., Brain Res., 712:117-121 (1996)). Interestingly, normal serum contains very low levels of soluble circulating MTf, but increased soluble serum MTf has been found in patients with advanced Alzheimer's Disease (Kennard, et al., Nat. Med., 2:1230-1235 (1996); U.S. Pat. No. 5,981,194).
The biochemical role and metabolism of MTf has proven difficult to elucidate. Based on appearances, MFt is deceptively similar to transferrin (Tf) and lactotransferrin (lactoferrin or Lf). In humans, these proteins share a 37-39% amino acid sequence homology. In particular, each of these proteins reversibly binds iron, and their N-terminal iron binding domains are quite similar (Baker, et al., TIBS, 12:350-353 (1987)).
However, functional parallels between these proteins have not been confirmed. For one thing, unlike Tf and Lf, MTf exists in both a membrane bound form and a serum soluble form. Further, in contrast to Tf and Lf, no cellular receptor for MTf has been identified. Serum soluble Tf is known to be taken into cells in an energy-dependent process mediated by the transferrin receptor (Tf-R) (Cook, et al., Annu. Rev. Med., 44:63-74) (1993)). Lf internalization is also likely to be mediated by a receptor mediated process (Fillebeen, et al., J. Biol. Chem., 274(11):701-7017 (1999)). Two known receptors for Lf are LRP-1 and RAGE, although others may exist (Melinger, et al., FEBS Letters, 360:70-74 (1995); Schmidt, J. Biol. Chem., 269(13):9882-9888 (1994).
With respect to the central nervous system (e.g., brain, spinal cord), there are at least three ways to enhance delivery: direct injection, permeabilization of the BBB, and modification of the drug. Direct injection involves injection of drug into brain tissue, bypassing the vasculature completely. This method suffers primarily from the risk of complications (infection, tissue damage) incurred by intra-cranial injections. This risk is compounded when considered in the context of a regular treatment regimen applied over the course of to the patient's life. It is also difficult, using a limited number of single site injections, to match the penetration that blood vessels (and hence, potentially, drug) have throughout the brain.
The second method entails non-specifically compromising the BBB with concomitant injection of intravenous drug. Permeabilization of the BBB is accomplished chemically. This method suffers from a lack of specificity. All those components in the blood that are necessarily excluded by the BBB will enter the brain along with the drug. The brain is left vulnerable under these conditions and damage would be anticipated over the course of a life-long regimen of treatment.
The third means of increasing brain availability of blood borne drug entails specific functionalization of the drug with moieties that facilitate transport through an uncompromised BBB. This method has the advantages of specific BBB infiltration and convenient intravenous administration. A method of increasing the ability of a therapeutic agent to cross the blood brain barrier is taught in U.S. Pat. No. 6,455,494, incorporated herein by reference its entirety, which discloses the use of p97 as a carrier for delivering a therapeutic drug across the blood brain barrier.
p97 (melanotransferrin) is a naturally occurring human protein. p97 was discovered and characterized as a cell surface marker for human melanoma (melanoma-associated antigen), but has more recently been found in other tumor types, as well as in normal human brain and liver tissue, in trace amounts in other body tissues and in serum. The role of p97 in the body is unknown, but based on its structure and binding properties, it is thought to be involved in the transport of metal ions (e.g. iron) into cells. Jefferies, et al. have been working with p97 since 1992 (U.S. Pat. No. 5,981,194). These investigations have focused on p97 as a diagnostic marker for Alzheimer's disease (AD). Synapse has developed a blood (serum) test for AD that is based on the finding that the p97 serum level increases with the progression of the disease. During the development of this test, it was discovered that p97 is actively transported from the blood into the brain tissue of normal individuals. This discovery was the impetus for the development of p97 as a potential transport system to deliver molecules from the blood, across the BBB to reach brain interstitial fluids.
The key event for the successful delivery of therapeutic agents into brain is the transport of these large molecules across the tight network of capillary endothelial cells that comprise the BBB. During the last few years, it has been demonstrated both in vitro and in animal models that small synthetic molecules, large glycoprotein enzymes, and large inorganic particles (5 nm colloidal gold particles) chemically linked to p97, can be transported across the BBB to brain cells. Such transport of large molecules across the BBB involves a process known as transcytosis. This is a mechanism whereby molecules are picked up from the blood and transported through the capillary cells of the otherwise intact BBB to the brain tissue.
Transcytosis pathways are distinct from other vesicular traffic within the capillary endothelial cell and transit occurs without alteration of the transported materials. Transcytosis is a cell-type specific process mediated by receptors on the BBB endothelial surface. The transport of p97-conjugates (i.e., p97 chemically linked to macromolecules) across the BBB occurs by transcytosis. p97 conjugated to the enzyme horseradish peroxidase (HRP) (an example of an enzyme “payload”) can be transported across the BBB.
For an effective treatment of LSDs, a therapeutic agent (e.g., the deficient enzyme, or another enzyme or protein having a desired therapeutic or missing enzyme activity) must be taken up by the affected cells and routed to the lysosome where it acts upon the excessive or harmful amount of storage material residing therein. Applicants provide below compositions and methods for treating LSDs involving the use of p97 proteins to target delivery of therapeutic agents, including proteins or enzymes deficient in LSDs, to the lysosomes of cells.